An acytokinetic cell division creates PIP2-enriched membrane asymmetries leading to slit diaphragm assembly in Drosophila nephrocytes.

Vertebrate podocytes and Drosophila nephrocytes display slit diaphragms, specialised cell junctions that are essential for the execution of the basic excretory function of ultrafiltration. To elucidate the mechanisms of slit diaphragm assembly we have studied their formation in Drosophila embryonic garland nephrocytes. These cells of mesenchymal origin lack overt apical-basal polarity. We find that their initial membrane symmetry is broken by an acytokinetic cell division that generates PIP2-enriched domains at their equator. The PIP2-enriched equatorial cortex becomes a favourable domain for hosting slit diaphragm proteins and the assembly of the first slit diaphragms. Indeed, when this division is either prevented or forced to complete cytokinesis, the formation of diaphragms is delayed to larval stages. Furthermore, although apical polarity determinants also accumulate at the equatorial cortex, they do not appear to participate in the recruitment of slit diaphragm proteins. The mechanisms we describe allow the acquisition of functional nephrocytes in embryos, which may confer on them a biological advantage similar to the formation of the first vertebrate kidney, the pronephros.

Phospholipid scramblase 1: an essential component of the nephrocyte slit diaphragm.

Blood ultrafiltration in nephrons critically depends on specialized intercellular junctions between podocytes, named slit diaphragms (SDs). Here, by studying a homologous structure found in Drosophila nephrocytes, we identify the phospholipid scramblase Scramb1 as an essential component of the SD, uncovering a novel link between membrane dynamics and SD formation. In scramb1 mutants, SDs fail to form. Instead, the SD components Sticks and stones/nephrin, Polychaetoid/ZO-1, and the Src-kinase Src64B/Fyn associate in cortical foci lacking the key SD protein Dumbfounded/NEPH1. Scramb1 interaction with Polychaetoid/ZO-1 and Flotillin2, the presence of essential putative palmitoylation sites and its capacity to oligomerize, suggest a function in promoting SD assembly within lipid raft microdomains. Furthermore, Scramb1 interactors as well as its functional sensitivity to temperature, suggest an active involvement in membrane remodeling processes during SD assembly. Remarkably, putative Ca2+-binding sites in Scramb1 are essential for its activity raising the possibility that Ca2+ signaling may control the assembly of SDs by impacting on Scramb1 activity.

Endocytosis mediated by an atypical CUBAM complex modulates slit diaphragm dynamics in nephrocytes.

The vertebrate endocytic receptor CUBAM, consisting of three cubilin monomers complexed with a single amnionless molecule, plays a major role in protein reabsorption in the renal proximal tubule. Here, we show that Drosophila CUBAM is a tripartite complex composed of Amnionless and two cubilin paralogues, Cubilin and Cubilin2, and that it is required for nephrocyte slit diaphragm (SD) dynamics. Loss of CUBAM-mediated endocytosis induces dramatic morphological changes in nephrocytes and promotes enlarged ingressions of the external membrane and SD mislocalisation. These phenotypes result in part from an imbalance between endocytosis, which is strongly impaired in CUBAM mutants, and exocytosis in these highly active cells. Of note, rescuing receptor-mediated endocytosis by Megalin/LRP2 or Rab5 expression only partially restores SD positioning in CUBAM mutants, suggesting a specific requirement of CUBAM in SD degradation and/or recycling. This finding and the reported expression of CUBAM in podocytes suggest a possible unexpected conserved role for this endocytic receptor in vertebrate SD remodelling.

Drosophila Lipophorin Receptors Recruit the Lipoprotein LTP to the Plasma Membrane to Mediate Lipid Uptake.

Lipophorin, the main Drosophila lipoprotein, circulates in the hemolymph transporting lipids between organs following routes that must adapt to changing physiological requirements. Lipophorin receptors expressed in developmentally dynamic patterns in tissues such as imaginal discs, oenocytes and ovaries control the timing and tissular distribution of lipid uptake. Using an affinity purification strategy, we identified a novel ligand for the lipophorin receptors, the circulating lipoprotein Lipid Transfer Particle (LTP). We show that specific isoforms of the lipophorin receptors mediate the extracellular accumulation of LTP in imaginal discs and ovaries. The interaction requires the LA-1 module in the lipophorin receptors and is strengthened by a contiguous region of 16 conserved amino acids. Lipophorin receptor variants that do not interact with LTP cannot mediate lipid uptake, revealing an essential role of LTP in the process. In addition, we show that lipophorin associates with the lipophorin receptors and with the extracellular matrix through weak interactions. However, during lipophorin receptor-mediated lipid uptake, LTP is required for a transient stabilization of lipophorin in the basolateral plasma membrane of imaginal disc cells. Together, our data suggests a molecular mechanism by which the lipophorin receptors tether LTP to the plasma membrane in lipid acceptor tissues. LTP would interact with lipophorin particles adsorbed to the extracellular matrix and with the plasma membrane, catalyzing the exchange of lipids between them.

Drosophila lipophorin receptors mediate the uptake of neutral lipids in oocytes and imaginal disc cells by an endocytosis-independent mechanism.

Lipids are constantly shuttled through the body to redistribute energy and metabolites between sites of absorption, storage, and catabolism in a complex homeostatic equilibrium. In Drosophila, lipids are transported through the hemolymph in the form of lipoprotein particles, known as lipophorins. The mechanisms by which cells interact with circulating lipophorins and acquire their lipidic cargo are poorly understood. We have found that lipophorin receptor 1 and 2 (lpr1 and lpr2), two partially redundant genes belonging to the Low Density Lipoprotein Receptor (LDLR) family, are essential for the efficient uptake and accumulation of neutral lipids by oocytes and cells of the imaginal discs. Females lacking the lpr2 gene lay eggs with low lipid content and have reduced fertility, revealing a central role for lpr2 in mediating Drosophila vitellogenesis. lpr1 and lpr2 are transcribed into multiple isoforms. Interestingly, only a subset of these isoforms containing a particular LDLR type A module mediate neutral lipid uptake. Expression of these isoforms induces the extracellular stabilization of lipophorins. Furthermore, our data indicate that endocytosis of the lipophorin receptors is not required to mediate the uptake of neutral lipids. These findings suggest a model where lipophorin receptors promote the extracellular lipolysis of lipophorins. This model is reminiscent of the lipolytic processing of triglyceride-rich lipoproteins that occurs at the mammalian capillary endothelium, suggesting an ancient role for LDLR-like proteins in this process.

Uptake of the necrotic serpin in Drosophila melanogaster via the lipophorin receptor-1.

The humoral response to fungal and Gram-positive infections is regulated by the serpin-family inhibitor, Necrotic. Following immune-challenge, a proteolytic cascade is activated which signals through the Toll receptor. Toll activation results in a range of antibiotic peptides being synthesised in the fat-body and exported to the haemolymph. As with mammalian serpins, Necrotic turnover in Drosophila is rapid. This serpin is synthesised in the fat-body, but its site of degradation has been unclear. By "freezing" endocytosis with a temperature sensitive Dynamin mutation, we demonstrate that Necrotic is removed from the haemolymph in two groups of giant cells: the garland and pericardial athrocytes. Necrotic uptake responds rapidly to infection, being visibly increased after 30 mins and peaking at 6-8 hours. Co-localisation of anti-Nec with anti-AP50, Rab5, and Rab7 antibodies establishes that the serpin is processed through multi-vesicular bodies and delivered to the lysosome, where it co-localises with the ubiquitin-binding protein, HRS. Nec does not co-localise with Rab11, indicating that the serpin is not re-exported from athrocytes. Instead, mutations which block late endosome/lysosome fusion (dor, hk, and car) cause accumulation of Necrotic-positive endosomes, even in the absence of infection. Knockdown of the 6 Drosophila orthologues of the mammalian LDL receptor family with dsRNA identifies LpR1 as an enhancer of the immune response. Uptake of Necrotic from the haemolymph is blocked by a chromosomal deletion of LpR1. In conclusion, we identify the cells and the receptor molecule responsible for the uptake and degradation of the Necrotic serpin in Drosophila melanogaster. The scavenging of serpin/proteinase complexes may be a critical step in the regulation of proteolytic cascades.

Patched, the receptor of Hedgehog, is a lipoprotein receptor.

The Hedgehog (Hh) family of secreted signaling proteins has a broad variety of functions during metazoan development and implications in human disease. Despite Hh being modified by two lipophilic adducts, Hh migrates far from its site of synthesis and programs cellular outcomes depending on its local concentrations. Recently, lipoproteins were suggested to act as carriers to mediate Hh transport in Drosophila. Here, we examine the role of lipophorins (Lp), the Drosophila lipoproteins, in Hh signaling in the wing imaginal disk, a tissue that does not express Lp but obtains it through the hemolymph. We use the up-regulation of the Lp receptor 2 (LpR2), the main Lp receptor expressed in the imaginal disk cells, to increase Lp endocytosis and locally reduce the amount of available free extracellular Lp in the wing disk epithelium. Under this condition, secreted Hh is not stabilized in the extracellular matrix. We obtain similar results after a generalized knock-down of hemolymph Lp levels. These data suggest that Hh must be packaged with Lp in the producing cells for proper spreading. Interestingly, we also show that Patched (Ptc), the Hh receptor, is a lipoprotein receptor; Ptc actively internalizes Lp into the endocytic compartment in a Hh-independent manner and physically interacts with Lp. Ptc, as a lipoprotein receptor, can affect intracellular lipid homeostasis in imaginal disk cells. However, by using different Ptc mutants, we show that Lp internalization does not play a major role in Hh signal transduction but does in Hh gradient formation.

Genome-wide phenotypic RNAi screen in the Drosophila wing: global parameters.

We have screened a collection of UAS-RNAi lines targeting 10,920 Drosophila protein-coding genes for phenotypes in the adult wing. We identified 3653 genes (33%) whose knockdown causes either larval/pupal lethality or a mutant phenotype affecting the formation of a normal wing. The most frequent phenotypes consist of changes in wing size, vein differentiation, and patterning, defects in the wing margin and in the apposition of the dorsal and ventral wing surfaces. We also defined 16 functional categories encompassing the most relevant aspect of each protein function and assigned each Drosophila gene to one of these functional groups. This allowed us to identify which mutant phenotypes are enriched within each functional group. Finally, we used previously published gene expression datasets to determine which genes are or are not expressed in the wing disc. Integrating expression, phenotypic and molecular information offers considerable precision to identify the relevant genes affecting wing formation and the biological processes regulated by them.

Specificity of Distalless repression and limb primordia development by abdominal Hox proteins.

In Drosophila, differences between segments, such as the presence or absence of appendages, are controlled by Hox transcription factors. The Hox protein Ultrabithorax (Ubx) suppresses limb formation in the abdomen by repressing the leg selector gene Distalless, whereas Antennapedia (Antp), a thoracic Hox protein, does not repress Distalless. We show that the Hox cofactors Extradenticle and Homothorax selectively enhance Ubx, but not Antp, binding to a Distalless regulatory sequence. A C-terminal peptide in Ubx stimulates binding to this site. However, DNA binding is not sufficient for Distalless repression. Instead, an additional alternatively spliced domain in Ubx is required for Distalless repression but not DNA binding. Thus, the functional specificities of Hox proteins depend on both DNA binding-dependent and -independent mechanisms.

A specific isoform of Pyd/ZO-1 mediates junctional remodeling and formation of slit diaphragms.

The podocyte slit diaphragm (SD), responsible for blood filtration in vertebrates, is a major target of injury in chronic kidney disease. The damage includes severe morphological changes with destabilization of SDs and their replacement by junctional complexes between abnormally broadened foot processes. In Drosophila melanogaster, SDs are present in nephrocytes, which filter the fly's hemolymph. Here, we show that a specific isoform of Polychaetoid/ZO-1, Pyd-P, is essential for Drosophila SDs, since, in pyd mutants devoid of Pyd-P, SDs do not form and the SD component Dumbfounded accumulates at ectopic septate-like junctions between abnormally aggregated nephrocytes. Reintroduction of Pyd-P leads to junctional remodeling and their progressive normalization toward SDs. This transition requires the coiled-coil domain of Pyd-P and implies formation of nonclathrin vesicles containing SD components and their trafficking to the nephrocyte external membrane, where SDs assemble. Analyses in zebrafish suggest a conserved role for Tjp1a/ZO-1 in promoting junctional remodeling in podocytes.

jing is required for wing development and to establish the proximo-distal axis of the leg in Drosophila melanogaster.

The establishment of the proximo-distal (PD) axis in the legs of Drosophila melanogaster requires the expression of a nested set of transcription factors that are activated in discreet domains by secreted signaling molecules. The precise regulation of these transcription factor domains is critical for generating the stereotyped morphological characteristics that exist along the PD axis, such as the positioning of specific bristle types and leg joints. Here we provide evidence that the Zn-finger protein encoded by the gene jing is critical for PD axis formation in the Drosophila legs. Our data suggest that jing represses transcription and that it is necessary to keep the proximal gene homothorax (hth) repressed in the medial domain of the PD axis. We further show that jing is also required for alula and vein development in the adult wing. In the wing, Jing is required to repress another proximal gene, teashirt (tsh), in a small domain that will give rise to the alula. Interestingly, we also demonstrate that two other genes affecting alula development, Alula and elbow, also exhibit tsh derepression in the same region of the wing disc as jing- clones. Finally, we show that jing genetically interacts with several members of the Polycomb (Pc) group of genes during development. Together, our data suggest that jing encodes a transcriptional repressor that may participate in a subset of Pc-dependent activities during Drosophila appendage development.

Distinct functions of homeodomain-containing and homeodomain-less isoforms encoded by homothorax.

The homothorax (hth) gene of Drosophila melanogaster is required for executing Hox functions, for head development, and for forming the proximodistal (PD) axis of the appendages. We show that alternative splicing of hth generates two types of protein isoforms, one that contains a DNA-binding homeodomain (HthFL) and one that does not contain a homeodomain (HDless). Both types of Hth isoforms include the evolutionarily conserved HM domain, which mediates a direct interaction with Extradenticle (Exd), another homeodomain protein. We show that although both HthFL and HDless isoforms of Hth can induce the nuclear localization of Exd, they carry out distinct sets of functions during development. Surprisingly, we find that many of hth's functions, including PD patterning and most Hox-related activities, can be executed by the HDless isoforms. In contrast, antennal development shows an absolute dependency on the HthFL isoform. Thus, alternative splicing of hth results in the generation of multiple transcription factors that execute unique functions in vivo. We further demonstrate that the mouse ortholog of hth, Meis1, also encodes a HDless isoform, suggesting that homeodomain-less variants of this gene family are evolutionarily ancient.

Boca, an endoplasmic reticulum protein required for wingless signaling and trafficking of LDL receptor family members in Drosophila.

The maturation of cell surface receptors through the secretory pathway often requires chaperones that aid in protein folding and trafficking from one organelle to another. Here we describe boca, an evolutionarily conserved gene in Drosophila melanogaster, which encodes an endoplasmic reticulum protein that is specifically required for the intracellular trafficking of members of the low-density lipoprotein family of receptors (LDLRs). Two LDLRs in flies, Arrow, which is required for Wingless signal transduction, and Yolkless, which is required for yolk protein uptake during oogenesis, both require boca function. Consequently, boca is an essential component of the Wingless pathway but is more generally required for the activities of multiple LDL receptor family members.

Boca-dependent maturation of beta-propeller/EGF modules in low-density lipoprotein receptor proteins.

The extracellular portions of cell surface receptor proteins are often comprised of independently folding protein domains. As they are translated into the endoplasmic reticulum (ER), some of these domains require protein chaperones to assist in their folding. Members of the low-density lipoprotein receptor (LDLR) family require the chaperone called Boca in Drosophila or its ortholog, Mesoderm development, in the mouse. All LDLRs have at least one six-bladed beta-propeller domain, which is immediately followed by an epidermal growth factor (EGF) repeat. We show here that Boca is specifically required for the maturation of these beta-propeller/EGF modules through the secretory pathway, but is not required for other LDLR domains. Protein interaction data suggest that as LDLRs are translated into the ER, Boca binds to the beta-propeller. Subsequently, once the EGF repeat is translated, the beta-propeller/EGF module achieves a more mature state that has lower affinity for Boca. We also show that Boca-dependent beta-propeller/EGF modules are found not only throughout the LDLR family but also in the precursor to the mammalian EGF ligand.